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A catalogue record for this book is available from the British Library.Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not beavailable in electronic books.Cover image: Photos by Daniel Merrifield.Set in 10/12pt Times by Laserwords Private Limited, Chennai, India

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Contents

List of ContributorsPreface1 The Gastrointestinal Tract of FishArun Kumar Ray and Einar Ringø1.11.21.31.41.51.61.71.81.9

IntroductionBacterial aspects of live feedBacterial control of live feed culturesEnrichment of live feed and microbial implicationsProbiotics in live feed productionBioencapsulation of probiotics in live food and delivery to larvaePrebiotics and synbiotics in live feedConclusions and future perspectivesReferences

Since the initial investigations on the gut microbiota of fish some five decades ago, considerable information has been presented on their composition, abundance, diversity and activity.Numerous studies have demonstrated that these communities are complex and generally of lowcultivability, containing Bacteria, Archaea, viruses, yeasts and protists. However, little attention has been paid to the Archaea, protists or viruses but several studies have revealed diversecommunities of bacteria and yeast. These microbes have major implications on host health,development, welfare and nutrition and therefore great efforts have been made in the past twodecades to fortify these communities and maintain microbial balance. Among such efforts theapplications of probiotics and prebiotics have been at the forefront. The scientific evidencewhich underpins the efficacy, and to some extent elucidates their modes of action, has beencomprehensive, although not always reproducible. This body of evidence has helped to create amarket and drive demand for commercial probiotics and prebiotics for use in aquaculture operations globally. As such, many feed manufacturers, multi-nationals and small domestic operations, routinely add pro- and prebiotic products to their feed formulations. The extent of theireconomic benefits is not yet clear, as such information is not often openly discussed by fishfarmers, but the increasing demand and increasing volumes of probiotic/prebiotic aquafeedsproduced are positive indicators for industrial level applications. Future research efforts shouldfocus on better understanding of the modes of action, which must include a better understanding of the composition and activity of indigenous microbiomes, as well as the effects on the hostitself, so that optimisation of probiotic/prebiotic selection, dosage and application strategiescan occur.The chapters within this book address these issues and are advised reading for an understanding of the historical development of these products, their known mechanisms of actionand their degree of efficacy as presently demonstrated in the literature. We also hope thatthe fundamental material provided on the gut microbiota itself, and more broad aspects ofmicrobe-live feed interactions, are useful reading for researchers, academics and students. Wewish to thank the authors that have contributed to this book, as well as our PhD students andpost-doctoral staff whom have also assisted in the collection of data and literature. We are alsograteful to the assistance of the production staff at Wiley-Blackwell for their support.Daniel Merrifield and Einar Ringø

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The Gastrointestinal Tract of Fish

Arun Kumar Ray1 and Einar Ringø21 Department2 Norwegian

of Zoology, Visva-Bharati University, West Bengal, IndiaCollege of Fishery Science, UiT The Arctic University of Norway

ABSTRACTThe organization of the gastrointestinal (GI) tract of fish follows the basic features as in othervertebrate groups with a degree of variation in phylogeny and ontogeny, feeding habits, diet,nutrition, physiological conditions and the special functions the gut may perform. There areenormous variations in the morphology of the GI tract among various fish species. The variations in the organization of the GI tract ensure optimum utilization of dietary nutrients, whichin many cases means efficient primary digestion and a large intestinal absorptive surface area.Different fish species have adapted different approaches to accommodate this objective. Ofparticular interest to fish nutritionists is the comparison of morphological features in relationto natural diets. In order to compare data obtained from one fish species with other species, itis essential to make divisions into a broad line of common morphological features.

1.1 INTRODUCTIONDetailed descriptions of the anatomy and physiology of GI tracts of numerous fish species havebeen covered in several reviews (Suyehiro 1942; Barrington 1957; Kapoor et al. 1975; Harder1975; Fänge and Grove 1979; Smith 1989; Stevens 1988; Olsen and Ringø 1997; Wilson andCastro 2011). Fish have the ability to rapidly and reversibly adapt GI tract characteristics tomatch the changes in functional demands that occur during their life history (e.g. metamorphosis, anadrome or catadrome migrations) or more frequently (day-to-day or seasonal shifts indiet or environmental conditions); this ability is dependent on endocrine signalling pathwayswhich are augmented by the enteric nervous system (Karila et al. 1998). The wide diversity andlevels of hormones and signalling molecules secreted by the numerous types of GI tract andendocrine pancreas cells allow fish to rapidly and reversibly alter characteristics of the GI tract

and other organ systems to adapt to changes in the contents of the GI tract (amounts and typesof nutrients, pH, ionic composition etc.) and environmental conditions (Holst et al. 1996).The key feature of the alimentary tract is its ability to digest foodstuffs to make themsuitable for absorption by various transport mechanisms in the wall compartments of different GI sections (Bakke et al. 2011). Besides the hydrolytic reactions catalysed by endogenousenzymes secreted by the pancreas and cells in the gut wall, which are considered to play themajor roles in digestion, fermentation plays key roles in digestive processes in many monogastrics. The role of fermentation in fish is less clear due to a lack of knowledge, but it isconsidered to be of minor quantitative importance for nutrient supply in cold water species.However, qualitative importance may be significant regarding specific nutrients and immunestimulating processes.The anatomy and physiology of the GI tract are important determinants for the establishment and for the quantitative as well as the qualitative aspects of its microbiota. Themicrobial communities may seem to be assembled in predictable ways (Rawls et al. 2006). Inthis study the authors showed that microbial communities transplanted from mice to gnotobiotic zebrafish (Danio rerio) alter quantitatively in the direction of the normal biota of thezebrafish species and vice versa. This indicates that environmental conditions of the intestine,determined by species-specific parameters along the GI tract such as anatomy, endogenousinputs of digestive secretions, pH, osmolality, redox potential, compartment size and structure,passage rate and residence time, help to define and shape the GI tract microbiota. However,diet composition is also an important environmental condition for fish development. Diet composition is ideally species specific regarding available essential nutrients, but supplies variableamounts of unavailable material depending on the feedstuffs used in the diet formulations. Thegut microbiota is also probably inevitably linked to digestion by the production of exogenousenzymes and vitamins produced which might aid host digestive function (Ray et al. 2012).This chapter summarizes the current state of knowledge highlighting the morphological andhistological variations in the lower GI tract of fish associated with digestion and absorption;comprehensive reviews on the gut microbiota are presented in Chapters 4–6.

1.2 ANATOMY OF GI TRACTThe structure and functional characteristics of the GI tract vary widely among species(Suyehiro 1942) and seem, to a great extent, to match the wide diversity of feeding habitsand environmental conditions exploited by fish. The structure of the alimentary canal variesin different species of fish, and is generally adapted in relation to the food and feeding habits.Depending on feeding habits and diet, fish are generally classified as carnivorous (eatingfish and larger invertebrates), herbivorous (consuming mainly plant material), omnivorous(consuming a mixed diet) and detritivorous (feeding largely on detritus) (De Silva andAnderson 1995; Olsen and Ringø 1997; Ringø et al. 2003), together with the genera Panaqueand Chochliodon which are capable of digesting wood. However, such division may notalways be correct since most species consume mixed diets or their feeding habits may changethrough the life cycle (Olsen and Ringø 1997). The variation becomes obvious by comparingthe GI tract characteristics of carnivorous and herbivorous fish and those from freshwaterand seawater. The mucosal lining of the GI tract represents an interface between the externaland internal environments, and in conjunction with the associated organs (e.g. pancreas, liver

and gall bladder) provides the functions of digestion, osmoregulation, immunity, endocrineregulation of GI tract and systemic functions, and elimination of environmental contaminantsand toxic metabolites. The GI tract is basically a tube that courses through the body. The GItract in Atlantic cod (Gadus morhua L.) is shown in Figure 1.1. This tract is divided into thefollowing characteristic regions: mouth, gill arch, oesophagus, stomach, mid intestine, distalintestine and fermentation chamber.

1.3 STOMACH AND INTESTINAL BULBTwo main groups of fish are commonly distinguished on the basis of presence or absence ofstomach. The most remarkable feature of the digestive system of lampreys, hagfish, chimaeras,and many herbivorous fishes belonging to Cyprinidae, Cyprinodontidae, Balistidae, Labridae,Scomberesocidae and Scaridae, is the lack of a true stomach. In cyprinids, for example mrigal(Cirrhinus mrigala), the anterior part of the intestine becomes swollen to form a sac-like structure called the intestinal bulb or pseudogaster (Figure 1.2). In the absence of a stomach, theanterior intestine performs the function of temporary storage of ingested food (Sinha 1983). Instomachless fish the intestinal bulb apparently secretes mucus, and histologically the mucosaresembles closely that of the intestine and is devoid of any digestive components (Horn et al.2006; Manjakasy et al. 2009). The mucosa of the intestinal bulb is thrown into prominent foldsor villi (for lack of a better term; strictly speaking they are not true villi due to the absenceof lacteals) that are lined with absorptive and mucus-secreting cells. The absence of stomachin many stomachless fish is compensated by the presence of pharyngeal teeth or gizzards forgrinding food (Suyehiro 1942; Fänge and Grove 1979). Wood-eating fishes have specificallyadapted spoon-shaped teeth for efficiently rasping wood (Nelson et al. 1999). The lack of astomach in some species of fish raises questions regarding its significance. Several hypotheseshave been put forward to explain the absence of a stomach which are often contradictory andspeculative (for review, see Wilson and Castro 2011). The shape, size and structure of thestomach, when present, are related to the duration between meals and the nature of the diet(Suyehiro 1942; Smith 1989; De Silva and Anderson 1995). A stomach is defined as a portion

of the digestive tract with distinctive cell lining, where acid is secreted, usually along with somedigestive enzymes like pepsin (Olsen and Ringø 1997). In his early study, Suyehiro (1942)classified stomachs of fish into five categories according to their morphological appearance:(a) straight tube (Pleuronectidae, Esox), (b) U-shape (Salmonids), (c) V-shape (Plecoglossidae, Mugilidae, Salmonidae, Sparidae), (d) Y-shape (Mugilidae, Clupeidae), and (e) I-shape(Carangidae, Gadidae, Scombridae, Serranidae). The highest degree of modifications of thepyloric stomach have been reported in several members of Clupeoidei, Channidae, Mugilidae, Acipenseridae, Coregoninae and Chanidae (milkfish, Chanos chanos) where it acts as a‘gizzard’ for trituration and mixing (Fänge and Grove 1979; Kapoor et al. 1975; Buddington1985; De Silva and Anderson 1995). This development of a ‘gizzard’ has been attributed tomicrophagy, and is thought to partly compensate for poor dentition (Pillay 1953). The anterior part of the stomach (cardiac or fundic region) is characterized by the presence of gastricglands (Figure 1.3A) and the musculature is also usually more prominent (De Silva and Anderson 1995). The stomach mucosa is lined with columnar epithelium and studded with minutedepressions, the gastric crypts or pits that lead into the tubular or alveolar gastric glands. Gastric glands are present in abundance throughout the cardiac stomach, so much so that theyoccupy the entire mucosal layer beneath the superficial epithelium (Figure 1.3A). This partof the stomach is secretory in nature and is responsible for storage and initial physical andenzymatic breakdown of the diet; readers with special interest in this topic are referred to thecomprehensive review of Bakke et al. (2011). The mucosa of the posterior part of the stomach (pyloric stomach) contains many mucus-producing tubular mucus glands or pyloric glands(Figure 1.3B). The number of these glands decreases considerably in the middle region andthey are completely absent in the posterior region. The pyloric stomach is completely devoid

In a number of fish species, several finger-like outgrowths develop from the anterior part of theintestine in the region of pylorus. These are called pyloric caeca or intestinal caeca, and openinto the lumen of the intestine. They are located proximal in the midgut region, and, when

present, number from a few as in murrel Channa punctatus (Figure 1.4) to several hundredas in Atlantic cod (Figure 1.1). The caeca of different species vary considerably in size, stateof branching and connection to the gut (Suyehiro 1942; Olsen and Ringø 1997; Ringø et al.2003). Histologically, they closely resemble the intestine (Figure 1.3D), and possibly serve toincrease the absorptive surface of the gut (Bergot et al. 1975). The pyloric caeca are alwaysabsent in stomachless fish (Barrington 1957; Kapoor et al. 1975). Although the presence orabsence of the pyloric caeca has no apparent correlation with the nature of the food or withfeeding habits (Khanna 1961; Mohsin 1962), the caeca are typically absent or much reducedin omnivorous and herbivorous species (Rust 2002). There is also no clear correlation betweenthe number of caeca and the length of the gut, and feeding habits (Harder 1975; Hossain andDutta 1996). Pyloric caeca have been reported to increase the surface area for digestion andabsorption but do not have any role in fermentation or storage (Buddington and Diamond1987). In salmonids, the pH of caeca and caecal intestine is 7.0 and 7.5, respectively (Ringøet al. 2003). Compared to the numerous studies evaluating the finfish gut microbiota (e.g.Cahill 1990; Ringø et al. 1995; Ringø and Gatesoupe 1998; Hansen and Olafsen 1999; Ringøand Birkbeck 1999; Austin 2006; Kim et al. 2007; Merrifield et al. 2011; Lauzon and Ringø2012), fewer studies have investigated the microbiota of pyloric caeca (Lesel and Pointel 1979;Gildberg et al. 1997; Gildberg and Mikkelsen 1998; Navarrete et al. 2009; Zhou et al. 2009b).

1.5 INTESTINEIn fish, the intestine is the main organ for digestion/absorption. In addition to digesting andabsorbing feedstuffs, the intestine is critical for water and electrolyte balance, endocrine regulation of digestion and metabolism, and immunity (Ringø et al. 2003). The intestine showsconsiderable variation in its length and arrangement in different species of fish (Kapoor et al.1975; Fänge and Grove 1979; Stevens 1988). Some fish have a relative intestinal length (RIL= length of intestine/length of body) less than 1, while some fish species have an RIL of 10to 20 times their body length (Suyehiro 1942; Olsen and Ringø 1997). The highest RIL generally occurs in herbivorous and detritivorous species (Figure 1.2), while the lowest is foundin strictly carnivorous and predatory species (Figures 1.1 and 1.4). The intestine in Cyprinidsand Loricariids exhibits a wide range of looping and coiled arrangements (Figure 1.5), while